Methods of capturing cell-free methylated DNA and uses of same

Patent metadata

Field	Value
Publication number	US-11078475-B2
Application number	US-201716098620-A
Country	US
Kind code	B2
Filing date	May 3, 2017
Priority date	May 3, 2016
Publication date	Aug 3, 2021
Grant date	Aug 3, 2021

Abstract

There is described herein, a method of capturing cell-free methylated DNA from a sample having less than 100 mg of cell-free DNA, comprising the steps of: subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA; adding a first amount of filler DNA to the sample, wherein at least a portion of the filler DNA is methylated; denaturing the sample; and capturing cell-free methylated DNA using a binder selective for methylated polynucleotides.

First claim

The invention claimed is:

1. A method of capturing cell-free methylated DNA from a sample having less than 100 ng of cell-free DNA, comprising the steps of:
a. subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA;
b. adding a first amount of filler DNA to the sample, wherein at least a portion of the filler DNA is methylated;
c. denaturing the sample; and
d. capturing cell-free methylated DNA using a binder selective for methylated polynucleotides;
wherein the cell-free DNA from the sample and the first amount of filler DNA together comprises at least 50 ng of total DNA.

2. The method of claim 1 further comprising the step of amplifying and subsequently sequencing the captured cell-free methylated DNA.

3. The method of claim 1 , wherein the sample has less than 50 ng of cell-free DNA.

4. The method of claim 1 , wherein the first amount of filler DNA comprises about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% methylated filler DNA with any remainder being unmethylated filler DNA.

5. The method of claim 1 , wherein the first amount of filler DNA is from 20 ng to 100 ng.

6. The method of claim 1 , wherein the cell-free DNA from the sample and the first amount of filler DNA together comprises at least 100 ng of total DNA.

7. The method of claim 1 , wherein the filler DNA is 50 bp to 800 bp long.

8. The method of claim 1 , wherein the filler DNA is double stranded.

9. The method of claim 1 , wherein the filler DNA is junk DNA.

10. The method of claim 1 , wherein the filler DNA is endogenous or exogenous DNA.

11. The method of claim 10 , wherein the filler DNA is non-human DNA.

12. The method of claim 1 , wherein the filler DNA is λ, DNA.

13. The method of claim 1 , wherein the binder is a protein comprising a Methyl-CpG-binding domain.

14. The method of claim 13 , wherein the protein is a MBD2 protein.

15. The method of claim 1 , wherein step (d) comprises immunoprecipitating the cell-free methylated DNA using an antibody.

16. The method of claim 15 , comprising adding at least 0.05 μg of the antibody to the sample for immunoprecipitation.

17. The method of claim 15 , wherein the antibody is 5-MeC antibody or 5-hydroxymethyl cytosine antibody.

18. The method of claim 15 , further comprising the step of adding a second amount of control DNA to the sample after step (b) for confirming the immunoprecipitation reaction.

19. The method of claim 1 , further comprising the step of adding a second amount of control DNA to the sample after step (b) for confirming the capture of cell-free methylated DNA.

20. A method of measuring a DNA methylation profile within the sample comprising the method of claim 1 .

21. A method of monitoring immune therapy or determining cell turnover comprising the method of claim 20 .

22. The method of claim 4 , wherein the first amount of filler DNA comprises between 5% and 50%, between 10% and 40%, or between 15% and 30% methylated filler DNA.

23. The method of claim 5 , wherein the first amount of filler DNA is from 30 ng to 100 ng, or from 50 ng to 100 ng.

Assignees

• Univ Health Network
• Sinai Health Sys

Inventors

• Diniz De Carvalho Daniel
• Shen shu yi
• Singhania Rajat

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q2600/154

  Methylation markers · CPC title

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title